Billie-Jo M. Kerns

Overexpression of p53 and HER-2/ neu proteins as prognostic markers in early stage breast cancer

ObjectiveOverexpression of the p53 and HER-2/neu oncogenes are the two most common genetic abnormalities associated with breast cancer. Shorter survival time has been reported in patients with tumors with p53 or HER-2/neu. This report analyzes a retrospective cohort of early stage breast cancers for both oncogenes and relates overexpression to clinicopathologic parameters and survival. MethodsImmunostaining for p53 and HER-2/neu was performed on 230 paraffin-embedded specimens of stage I and II breast cancers diagnosed and treated at Duke University Medical Center between 1984 and 1987. Positive staining for both p53 and HER-2/neu in paraffin-embedded tissues indicates an underlying genetic abnormality: point mutations in the p53 gene and amplification of the HER-2/neu gene. ResultsIn this cohort of patients, 24% were positive for p53 and 17% for HER-2/neu. Four per cent were positive for both oncogenes. Significant correlations were found between p53 immunostaining and increasing tumor size, stage, and low estrogen and progesterone receptor contents. Univariate analysis showed that p53 and HER-2/neu were indicators of overall and failure-free survival. An additive effect on survival was observed in patients with both oncogene abnormalities. Nodal status, HER-2/neu, and p53 all attained independent prognostic value in a multivariate analysis. ConclusionsThe p53 and HER-2/neu oncogenes have proven but limited prognostic value. An approach that combines several molecular genetic markers with established pathologic criteria may help physicians to make more accurate predictions of prognosis in patients with early stage breast cancer.

Differential Immunoreactivity of Epidermal Growth Factor Receptor in Benign, Dysplastic and Malignant Prostatic Tissues

To investigate epidermal growth factor receptor (EGFr) presence in the prostate, monoclonal antibody (clone EGFR1) immunohistochemical examination of radical prostatectomy specimens was performed (n = 37). All prostatic specimens contained benign prostatic hyperplasia (BPH) and/or dysplasia (prostatic intraepithelial neoplasia or PIN), as well as prostatic carcinoma (CaP). Areas of dysplasia were further categorized as to the basal cell layer and the luminal cell area. BPH, PIN, and CaP tissues in each specimen were analyzed by a single observer and graded on a scale from 0-4+. Fifteen samples were also analyzed for EGFr content utilizing a Cell Analysis Systems (CAS 200) image cytometer. EGFr immunoreactivity of BPH basal cells was significantly higher than EGFr immunoreactivity in areas of CaP (p < 0.001). EGFr staining of BPH basal cells was also significantly higher than that seen in PIN luminal cells (p < 0.001). Immunoreactivity of EGFr in PIN basal cells was significantly higher than in PIN luminal cells (p < 0.001). EGFr staining of basal cells in BPH tissues was higher than that seen in the PIN basal cell layer but the difference was not statistically significant (p = 0.06). The amount of staining present in PIN luminal cells was also significantly greater than in CaP tissues (p = 0.002). Quantitative image analysis utilizing the CAS 200 image cytometer was performed on BPH and CaP areas exclusively. EGFr immunoreactivity in basal cells of the BPH tissues was significantly greater than that seen in CaP tissues (p < 0.001). The decreased EGFr immunoreactivity in CaP may reflect a differentiating role for EGFr in normal tissues. Loss of EGFr influence may be associated with an increased proliferative state in PIN and CaP. Destruction or alteration of the epidermal grwoth factor receptor by a protease, such as prostatic specific antigen, may also explain our findings. At the present time the meaning of the different amounts of EGFr in the various types of prostate tissues is unknown.

Maintenance of DNA content and erbB-2 alterations in intraductal and invasive phases of mammary cancer

SummaryDuctal carcinomain situ (intraductal carcinoma) of the breast is a commonly recognized and curable clinical entity. Patients with intraductal carcinoma are at risk to develop invasive breast cancer presumably due to a transition from the noninvasive to the invasive phase of growth. Primary breast malignancies commonly display bothin situ and invasive phases of growth in the same tumor. In the current study, DNA content and alterations in the erbB-2 (HER-2/neu) oncogene product were examined simultaneously in both growth phases of primary breast cancers by image analysis. DNA content in the intraductal and invasive components of primary breast cancers were virtually identical (r = 0.979, p < 0.001). Quantitative image analysis was used to measure erbB-2 expression and categories of expression were related to copy number of the erbB-2 gene. Expression of erbB-2 was similar in both growth phases and implies identity of the erbB-2 genotype. The identity of DNA content suggests that the noninvasive and invasive phases within a single breast cancer are highly related. It is likely that erbB-2 gene number remains the same during progression from intraductal to invasive disease.

c-erbB-2 Expression in Breast Cancer Detected by Immunoblotting and Immunohistochemistry

Evidence that the c-erbB-2 proto-oncogene is important in prognosis and oncogenesis in a number of human malignancies is increasing. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized to determine the amplification and protein expression of this proto-oncogene, respectively. These extraction techniques are often time consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Immunohistochemistry (IHC), on the other hand, is more often time and cost effective. In addition, IHC may offer enhanced sensitivity over extraction techniques because of the in situ nature of analysis. In data presented here, 71 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilution DNA hybridization and IHC, respectively. In 65 (92%) of 71 cases, high-level expression was associated with gene amplification, whereas moderate or low-level expression was associated with a normal diploid gene copy number. In five of the six discrepant cases, IHC predicted amplification which was not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. In 39 of the 71 total cases, Western immunoblotting was compared with IHC in the assessment of oncoprotein expression. Concordance was found in 33 (85%) of 39 cases. In four of the six discrepant cases, high levels of c-erbB-2 expression were demonstrated by IHC but not by immunoblotting. In these cases, intraductal disease and stroma-rich tumors again led to a relative paucity of neoplastic tissue within the specimens. We conclude that IHC offers a favorable alternative to either Southern analysis or Western immunoblotting in the assessment of c-erbB-2 gene copy number and expression levels of oncoprotein in human mammary carcinoma. Furthermore, IHC may prove advantageous to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.

Prognostic value of MIB‐1 in advanced ovarian carcinoma as determined using automated immunohistochemistry and quantitative image analysis

The monoclonal antibody MIB‐1 is an immunohistochemical marker reacting most strongly with cells in late S phase, G2, and M portions of the cell cycle. This antibody, reactive in formalin‐fixed, paraffin‐embedded tissue, allows the quantitation of a proliferation index (PI) in both current clinical cases and archival material using a computerized image analyzer (CIA).

Differential immunoreactivity of transforming growth factor alpha in benign, dysplastic and malignant prostatic tissues

Immunohistochemical examination of radical prostatectomy specimens from 57 patients was performed to determine the differential expression of transforming growth factor alpha in the human prostate. In addition, epidermal growth factor receptor (EGFr) immunoreactivity was assessed in each case. Stromal versus epithelial staining was determined for each histological subtype: benign prostatic hypertrophy (BPH), prostatic intra-epithelial neoplasia (PIN), and prostatic cancer (CaP) by a single pathologist reviewer. TGFa staining was predominant in stroma while EGFr was localized to the epithelial basal cell layer. Immunoreactivity of both TGFa (P = 0.002) and EGFr (P < 0.001) revealed a significant reduction in CaP compared to BPH or PIN. Autocrine stimulation of EGFr by TGFa or other unrecognized factors may be present in CaP. Conversely, altered stromal influence of CaP via TGFa may be present. These observations could form the basis for future cancer therapeutic strategies using antagonist factors.

Differential immunoreactivity of her-2/neu oncoprotein in prostatic tissues.

To investigate HER-2/neu oncoprotein immunoreactivity, monoclonal antibody TA1 immunohistochemical examination of flash-frozen radical prostatectomy specimens was performed (n = 35). All prostatic specimens contained benign prostatic hyperplasia (BPH) and/or prostatic intraepithelial neoplasia (PIN), as well as prostatic carcinoma (CaP). HER-2/neu oncoprotein immunoreactivity in BPH tissues was not significantly different than that for the PIN basal cell layer (P = 0.10) or for the PIN luminal cells (P = 0.17). There was significantly more HER-2/neu oncoprotein immunoreactivity in BPH than in areas of CaP (P < 0.001). There was no significant difference in the amount of immunoreactivity present in PIN basal cells when compared to the PIN luminal cells (P = 0.49). Both the PIN basal cells and luminal cells stained for the HER-2/neu oncoprotein to a higher degree than cells in the CaP areas (P < 0.001 in both cases). HER-2/neu oncoprotein immunoreactivity is present at a significantly higher degree in BPH and PIN than in malignant prostatic epithelium.

DNA Ploidy of Basal Cell Carcinoma Determined by Image Cytometry of Fresh Smears

Image analysis of nuclear DNA content (DNA ploidy) was performed on smears of basal cell carcinoma (BCC) obtained during Mohs microscopically‐controlled surgery from 51 tumors. DNA ploidy was compared with histologic growth pattern and the contour of the invading edge. There was a statistically significantly increased frequency of DNA aneuploidy in smears from BCC exhibiting partial or total diffuse (infiltrative and superficial multicentric) growth patterns (80%; 32 of 40) as compared to solely circumscribed growth patterns (0%; 0 of 11) (p< 0.001).

Determination of Proliferation Index By MIB‐1 Immunostaining in Early Stage Breast Cancer Using Quantitative Image Analysis

Abstract: Several clinicopathologic variables influence prognosis in breast cancer, including stage, histologic grade, nodal status, and tumor size. Multiple studies have shown an independent value of proliferation index as a prognostic variable for the stratification into favorable and unfavorable groups. The monoclonal antibody MIB‐1 reacts with the same antigen site, not epitope, as recognized by the Ki‐67 antibody. Like Ki‐67, MIB‐1 reacts with cells in the late G1, S, M and G2 phases of the cell cycle, but MIB‐1 has the advantage of reacting with formalin‐fixed, paraffin‐embedded material. The authors investigated the feasibility of using image analysis to quantitate the MIB‐1 antibody staining (proliferation index [PI]) and predict survival in a series of 230 patients with stage I and stage II breast cancer. In a univariate Cox regression model, larger values of MIB‐1 were related to shorter survival times (p < 0.001). Exploratory statistical procedures were used to categorize the patients into good, intermediate, and poor survival groups using the following proliferation indices as cut‐points: <5%, 5–11%, and >11 %, respectively. Higher clinical stage was associated with higher MIB‐1 values and shorter survival (p = 0.01, and p = 0.003, respectively). Tumor size (p = 0.02) and nodal status (p = 0.05) were also associated with higher values of MIB‐1. After adjusting for age, clinical stage, nodal status, and tumor size in a multivariate analysis, MIB‐1 retained its prognostic significance (p < 0.0001) when considered as either a continuous or categorical variable. There were no significant associations between MIB‐1 determined proliferation index and age (p = 0.54), histologic grade (p = 0.69), nuclear grade (p = 0.06) or the presence of vascular invasion (p =.66). There is a strong statistical relationship between cell proliferative activity, as determined by MIB‐1 expression, and survival in early stage breast cancer.

Detection of a Novel Marker in the Bronchial Secretions of Patients with Non-Small Cell Lung Cancer using the 4B5 Monoclonal Antibody

A murine monoclonal antibody designated 4B5 was raised against the high molecular weight fraction of pooled sputum from patients with non‐small cell lung cancer (NSCLC). Immunohistochemical staining indicated that 4B5 binds to histologically normal bronchial epithelium distant from tumor in 72% (39 of 54) of patients with NSCLC, but it binds to the primary cancer in only 13% (7 of 54) of the same patients. The antibody reacted less intensely with the bronchial epithelium in 16.6% (3 of 18) of autopsied patients without significant lung disease. The antigen recognized by 4B5 is a high molecular weight glycoprotein of more than 400 kilodaltons, judged by gel filtration and sodium dodecyl sulfate‐poly‐acrylamide gel electrophoresis and western blot analysis. Antigenic activity persisted after heating and resisted treatment with neuraminidase, but it was destroyed using protease and periodate. Multiple epitopes were present on each molecule recognized by 4B5. The determinants recognized by this antibody deserve additional study as possible markers of premalignant change in patients with NSCLC.